Method of improving immune function in mamals using lactobacillus reuteri strains

ABSTRACT

The invention herein is related to the use of  Lactobacillus reuteri  strains as immune enhancing agents, methods of improving immune-function in mammals using  Lactobacillus reuteri  strains in products containing cells of such strains and the products as such. These strains exhibit good toxin binding and neutralizing effect, and exhibit good CD4+ cell recruitment.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the use of Lactobacillus reuteri strains asimmune enhancing agents, and improved methods of selecting strains thatare most beneficial for this purpose.

2. Description of the Related Art

Probiotics containing a wide variety of different gastrointestinalmicroorganisms have been formulated, primarily because of the increasein antibiotic-resistant pathogens. Strains of a wide variety ofLactobacillus species, including L. reuteri have been used in probioticformulations. Lactobacillus reuteri is one of the naturally occurringinhabitants of the gastrointestinal tract of animals, and is routinelyfound in the intestines of healthy animals. It is known to haveantibacterial activity. See, for example U.S. Pat. Nos. 5,439,678,5,458,875, 5,534,253, 5,837,238, and 5,849,289. When L. reuteri cellsare grown under anaerobic conditions in the presence of glycerol, theyproduce the antimicrobial substance known as reuterin(β-hydroxy-propionaldehyde).

Immunomodulating activity has also been associated with L. reuteri. See,for example “Biotherapeutic effects of probiotic bacteria on candidiasisin immunodeficient mice” by Wagner R D, et al, Infect Immune October1997 65:4165-72; however, differences in efficacy exists between strainsand methods are needed to select the most effective strains, for examplethe method selecting strains recruiting CD4+ cells, provided in thisinvention.

Furthermore, although L. reuteri is known to be used as a generallybeneficial probiotic, previous work has only to some extent realized theimportance of utilizing best Lactobacillus strains that neutralizetoxins produced by these pathogens already present in thegastrointestinal tract. See, for example, “Removal of common Fusariumtoxins in vitro by strains of Lactobacillus and Propionibacterium” by.El-Nezami H S, et al, Food Addit Contam Jul. 19, 2002:680-6. Such toxinneutralization, including binding, is not only as reported important forameliorating the direct effects caused by these toxin producingpathogens but also in reducing the general burden on the immune-system,according to this invention.

There are many different causes of gastrointestinal problems caused bypathogenic microorganisms. For example, Helicobacteri pylori causesgastric and duodenal ulcers, gastric cancer, and gastricmucosa-associated lymphoid tissue lymphoma. Certain pathogenicEscherichia coli strains produce toxins such as the vero toxin (VT)produced by E. coli O157:H7, against which antibiotics are less and lesseffective.

It is therefore an object of the invention to provide a method ofimproving immune-function in mammals using Lactobacillus reuteri strainsdetected to be effective in both CD4+ cell recruitment and in toxinneutralization, and to provide methods of selecting suchimmune-improving L. reuteri strains, and to provide products containingsuch strains. It is a further object of this invention to provide amethod of utilizing culture supernatants of strains of L. reuterieffective in reducing the immune burden of these toxins.

Other objects and advantages will be more fully apparent from thefollowing disclosure and appended claims.

SUMMARY OF THE INVENTION

The invention herein is related to the use of Lactobacillus reuteristrains as immune enhancing agents, methods of improving immune-functionin mammals using Lactobacillus reuteri strains in products containingcells of such strains and the products as such. These strains areselected to exhibit good toxin binding and neutralizing; and to exhibitgood CD4+ recruitment. Other objects and features of the inventions willbe more fully apparent from the following disclosure and appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Confirmation of inhibitory ability against the binding of verocytotoxin(VT) and Gb₃ receptor in a culture supernatant of L. reuterithrough competitive ELISA. Each reacted as follows, on plates coatedwith Gb₃, followed by performing ELISA using mAb against VT.

Control: VT+tryptic soy broth(TSB)

VT+G: VT+250 mM glycerol solution

VT+LRS: VT+a culture supernatant of L. reuteri incubated in 250 mMglycerol solution

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

The present invention relates to the use of Lactobacillus reuteristrains that exhibit good toxin binding and neutralizing effect; andexhibit good CD4+ cell recruitment for the production of a compositionfor improving immune-function in mammals. It also relates to productscomprising such Lactobacillus reuteri strains and a method of improvingimmune-function in mammals using such Lactobacillus reuteri strains.Further, it provides methods of selecting such immune-improving L.reuteri strains.

Cytokines are immune system proteins that are biological responsemodifiers. They coordinate antibody and T cell immune systeminteractions, and amplify immune reactivity (10). Cytokines includemonokines synthesized by macrophages and lympho-kines produced byactivated T lymphocytes and natural killer (NK) cells. The CD4+ subsetin both human and mice is based on cytokine production and effectorfunctions. Th1 cells synthesize interferon-gamma (INF-γ), IL-2 and tumornecrosis factor (TNF). They are mainly responsible for cellular immunityagainst intracellular microorganisms and fordelayed-type-hypersensitivity reactions. They affect Immunoglobulin G 2a(IgG2a) synthesis and antibody dependent cell mediated cytotoxicity. TheINF-γ they produce activates macrophages and consequently phagocytosis.Th2 cells synthesize interleukin-4 (IL-4), interleukin-5 (IL-5),interleukin-6 (IL-6), interleukin-9 (IL-9), interleukin-10 (IL-10), andinterleukin-13 (IL-13). They induce IgE and Ig G1 antibody responses,and mucosal immunity by synthesis of mast cells and eosinophil growthand differentiation factors, and facilitation of IgA synthesis.

Included in the invention are methods with various example stepsconfirming: administration of the L.reuteri strain, analysis of thestrain and efficacy of the used strain in both CD4+ cell recruitment andtoxin neutralization. Data indicate that for example a tabletformulation containing L. reuteri gives similar gastrointestinalcolonization levels as with direct administration of cell cultures. TheL. reuteri colonization is related to ingestion of L. reuteri, and thewashout period (the time it takes for L. reuteri levels to drop topre-ingestion levels) is at least 28 days after administration of thetablet, showing that the invention herein is applicable for L.reutericell cultures as well as products formulated to contain such L.reutericultures.

The invention preferably relates to the use of products comprisingL.reuteri strains as a probioticum for prophylactic improvement of theimmune-function in mammals. Such products may be various foodstuffs suchas a dietary supplement, confectionery or tablets containing cells ofthe selected strain.

The L.reuteri strains according to the invention may also be used forthe preparation of a drug for treatment of various microorganisms thatproduce toxin, such as Echericia coli. Enterohemorrhagic E. Coli and VTtoxins may thus be treated.

According to the invention both cells of L.reuteri strains and a culturesupernatant thereof may be used for the production of a prophylactic,probiotic type and pharmaceutical composition.

According to the invention any The L.reuteri strain exhibiting good CD4+cell recruitment and/or good toxin binding may be used. That CD4+ cellsare present may be tested using antibodies against CD4 e.g. withimmunohistochemical (such as in example 5) or immunoflourescent methods.Toxin binding may be confirmed by bringing L.reuteri cells orsupernatant in contact with toxin and test for the difference inavailable toxin e.g. as is done in example 1.

The probiotic, prophylactic and pharmaceutical products according to theinvention may comprise additives and excipients acceptable fornutritious or pharmaceutical use.

The features of the present invention will be more clearly understood byreference to the following examples, which are not to be construed aslimiting the invention.

EXAMPLE 1 Vero Toxin Study

Strains of three lactic acid bacteria, L. reuteri ATCC 55730, L.bulgaricus, strain LB12, (CHR, Horsholm, Denmark), and L. casei, strain01, (CHR, Horsholm, Denmark), were employed in this experiment. L.reuteri was incubated in an aerotropic fixing condition at 37° C. for24-48 hours after inoculating in MRS broth (plus 20 mM glucose). In somecases, this initial incubation was followed by centrifugation at 2,500rpm for 30 minutes, washing with phosphate buffered saline (PBS) twiceto remove medium components, suspension in 250 mM glycerol solution,followed by incubation in an aerotropic fixing condition at 37° C. for 6hours. L. bulgaricus and L. casei were incubated on MRS plus 20 mMglucose (without glycerol) in an aerotropic fixing condition at 37° C.for 24-48 hours. Each test lactic acid bacterium was employed followingadjusting to 2 g/30 ml (dry weight), centrifuging at 2,500 rpm for 30minutes after incubation, retrieving the supernatant, adjusting the pHto 7.0 with NaOH to inoculate vero cells (see below), and filteringthrough a 2.0 μm filter. Glycerol solution and MRS broth adjusted to pH7.0 served as a control, following filtering through a 2.0 μm filter.

Vero cells (African Green Monkey kidney cells, ATCC—CCL81) wereincubated in a minimum essential medium (MEM, Sigma) supplemented with10% fetal bovine serum (FBS, Sigma) at 25 g/ml gentamycin (Sigma) at 37°C. in a 10% CO₂ incubator for 48 hours, and were used after confirmingmonolayer formation.

Escherichia coli 0157:H7 (ATCC 43894), which secretes both VT1 and VT2,was employed, following inoculating to tryptic soy broth (TSB),incubating for 24 hours while stirring in a shaking incubator at 37° C.,centrifuging at 2,500 rpm for 30 minutes, and filtering the culturesupernatant.

500 Vero cells (2×10⁵ cells/ml) were inoculated to a 96-well plate andincubated at 37° C. in a 10% CO₂ incubator for 48 hours to confirmformation of a monolayer, followed by carrying out the experiment usingthe following treatments: A: VT only (positive control); B: TSB(Escherichia coli 0157:H7 culture medium); C: MRS broth (test lacticacid bacteria); D: glycerol solution (L. reuteri culture medium); E:VT+MRS broth; F: VT+glycerol solution; G: VT+glycerol solution culturesupernatant of L. reuteri; H: VT+MRS broth culture supernatant of L.bulgaricus; I: VT+culture supernatant of L. bulgaricus; and J:VT+culture supernatant of L. casei. Treatments B. C and D are todetermine whether each culture fluid itself causes cytotoxicity to Verocells; and E and F are to determine whether culture media of test lacticacid bacteria have neutralizing capability against VT in themselves.Each culture supernatant of test lactic acid bacteria in G, H, I and Jwas subject to 2× serial dilution, after each of the diluted culturesupernatants of test lactic acid bacteria and VT were combined by 400ml+100 ml; 300 ml+200 ml; 200 ml+300 ml; and 100 ml+400 ml,respectively, and then incubated at 37° C. in a 10% CO₂ incubator for 18hours to determine whether a cytopathic effect (CPE) appeared.

300 mM neutralized semicarbazide hydrochloride (Sigma), which inhibitsreuterin production, was added to a culture fluid of L. reuteri in 250mM glycerol solution to restrain reuterin production, and thesupernatant collected as above. 100 μl Vero cells were inoculated to a96-well plate and incubated at 37° C. in a 10% CO₂ incubator for 24hours to examine whether monolayers formed, following by performing thefollowing treatments: A: VT only; B: VT+25 mM glycerol solution; C:VT+glycerol solution culture supernatant of L. reuteri; and D: VT+glycerol solution culture supernatant of L. reuteri incubated aftertreatment with reuterin inhibitor (see above). Each culture fluid of L.reuteri treated with or without glycerol solution and reuterin inhibitorin B, C and D, was combined with VT by 20+80; 30+70; 40+60; 50+50;60+40; 70+30; and 80+20 μl respectively, and then incubated at 37° C. ina 10% CO₂ incubator for 18 hours to examine whether a cyptopathic effectappeared. After microscopic observation, culture fluids were dyed withcrystal violet and their O.D. (optical density) values were read at 490nm.

96-well plates were coated with Gb3 (globotriaosylceramide) (Sigma)blocked with 5% bovine serum albumin (BSA) and reacted with L. reutericulture supernatant that was incubated in either VT or VT+250 mMglycerol solution. Afterward, a monoclonal antibody (mAb) against VT wasused as a primary antibody, to which horseradish peroxidase(HRP)-conjugated goat anti-mouse IgG was added, and, followingdevelopment of O-phenylene diamine, its optical density (O.D.) was readat 490 nm through an ELISA reader. This experiment confirmed thepresence of any material inhibiting the interaction between L. reuteriand the Gb₃ receptor.

From the results of interaction between VT and 250 mM glycerol solutionand between VT and a culture supernatant of L. reuteri incubated inglycerol solution after coating with Gb₃, it was ascertained that acombination of VT and a culture supernatant of L. reuteri yielded a lowO.D. value at significant levels compared with that of VT only, as inFIG. 1. This means that the presence of a material that inhibitedbinding of VT and the Gb₃ receptor in a culture supernatant of L.reuteri could be detected indirectly.

96-well plates were coated with L. reuteri culture supernatant which wasincubated in VT/VT+250 mM glycerol solution, blocked with 3% BSA andreacted with VT. Afterward, a monoclonal antibody against VT was used asa primary antibody, to which horseradish peroxidase (HRP)-conjugat4edgoat anti-mouse IgG (H+L) was added, and, following development ofO-phenylene daimine, its optical density (O.D.) was read at 490 nmthrough an ELISA reader. This experiment confirmed the presence of aninteractive material with VT in the L. reuteri supernatant

EXAMPLE 2 Investigation of Neutralizing Effect of Lactic Acid Bacteriaon Vero Cytotoxin (VT) I and II Secreted by E. coli O157:H7

When TSB, MRS broth and glycerol solution were added to Vero cells, acytopathic effect was not seen. In addition, when both VT and MRSbroth/glycerol solution were added to Vero cells, CPE was observed inVero cells, which proves that the culture fluids themselves lacked aneutralizing capability against VT.

When each culture supernatant of test lactic acid bacteria was subjectedto adjusting to pH 7.0, filtering and combining with VT, the resultsshown in Table 1 were found. For L. bulgaricus and L. casei, CPEappeared in the entire range of concentrations, while for L. reuteri,CPE did not appear in many of the glycerol supernatants, except with theratio of test lactic acid bacteria to VT of 4:1, where there was muchless CPE. Thus, there was a discernible neutralizing capability againstVT of the culture supernatant incubated in 250 mM glycerol solution. Forincubation in MRS broth (+20 mM glucose), CPE appeared at allconcentrations. TABLE 1 Comparison of inhibitory ability againstcytopathic effect(CPE) of vero cells by vero cytotoxin(VT) in culturesupernatants of L. reuteri, L. bulgaricus and L. casei serialdilution(×2) of lactic acid bacteria Lactic acid lactic acid bacteriaculture supernatants bacteria culture supernatants(μl) VT(μl) 0 1 2 3 4L. reuteri 400 100 − − − +^(a) +^(a) (G)* 300 200 − − +^(a) + + 200 300− +^(a) + + + 100 400 +^(a) +^(a) + + + L. reuteri 400 100 +^(a) + + + +(MRS)** 300 200 + + + + + 200 300 + + + + + 100 400 + + + + + L.bulgaricus 400 100 + + + + + 300 200 + + + + + 200 300 + + + + + 100400 + + + + + L. casei 400 100 + + + + + 300 200 + + + + + 200300 + + + + + 100 400 + + + + +*The culture supernatant obtained following incubating L. reuteri in 250mM glycerol solution**The culture supernatant obtained following incubating L. reuteri inMRS broth(adding 20 mM glucose)−: No CPE(cytopathic effects)+: CPE^(a)Mild CPE

The result of the competitive ELISA and the binding assay between VT andL. reuteri culture supernatant were subjected to Student's t test usinga Microcal Origin 6.1 (Microcal Software, Inc., Boston, Mass.) program.

EXAMPLE 3 Administration of L. reuteri to Subjects

In this example subjects were given two chewing tablets twice per day,each tablet containing 1×10⁸ CFU (colony-forming units) of L. reuteri(SD2112: ATCC 55730) to give a total daily dose of 4×10⁸ CFU L. reuteri.All other excipients used in the tablets were well-known and compliedwith international pharmacopoeias. The study was performed in two parts:a gastroscopy session with investigation of the upper gastro-intestinaltract, and an ileoscopy session with investigation of the distal smallbowel (details below). The exclusion criteria were: antibiotics takentwo weeks before and during the study; probiotics taken three weeksbefore and during the study, ongoing treatment with gastro intestinalrelated drugs, and severe organic disease with need of regular treatment(e.g., cancer). The protocol for patient treatment was approved by theDanish Ethical Committee and was in accordance with the declaration ofHelsinki. The study was done in Denmark.

The Wilcoxon signed-rank test was used to compare symptoms, blood testvalues, stool content of L. reuteri, and histological differences beforeand after intake of L. reuteri. P<0.05 was considered significant.

All subjects completed the study. In the gastroscopy session, tenhealthy volunteers, aged greater than 18 years, with normal eatinghabits, were studied. A gastroduodenoscopy was performed following anovernight fast on day 0, before intake of L. reuteri, and on day 28, atthe end of the study. Biopsies were taken from the corpus and the antrumof the stomach and from the third part of the duodenum, both for cultureof L. reuteri and for histological examination. The average size of thebiopsy was 33 mg. The biopsies were immediately placed in PBS (2:1vol/wt of sample), stored on ice and transported to Biogaia ABLaboratories in Lund, Sweden for analysis of Lactobacillus reutericounts. The time from the isolation of tissue to analysis was within 36hours.

In the ileoscopy session, nine subjects with ileostomy followingcolectomy for ulcerative colitis (3 subjects) or Crohn's disease (6subjects) were studied. The small bowel was without signs ofinflammation in all subjects. An ileoscopy was performed on day 0 andday 28. Biopsies were taken as above within the distal 20 cm of thesmall bowel for culture of L. reuteri as well as other lactobacilli andfor histological examination.

EXAMPLE 4 Analyses for Microorganisms

Stool samples were collected from each subject before intake of L.reuteri (day 0) and at the end of the study (day 28). Feces in ileoscopysubjects (explained below) were taken from the stoma. Seven volunteersfrom the gastroscopy session and four volunteers from the ileoscopysession collected stool samples at day 42 (14 days after cessation ofthe L. reuteri intake). The samples (no les than 5 g) were collectedinto a sterile container and placed immediately in a refrigerator.Within 24 hours, 20 ml (1:5, wt/vol) of 0.1% peptone water was added.The sample was homogenized and aliquots were dispensed into cryo-vialsbefore freezing immediately at −70° C. The samples were shipped, frozenon dry ice, to Biogaia AB laboratory for analysis of total Lactobacillusand L. reuteri. There, the samples were thawed, diluted and plated onMRS-3 agar containing vancomycin (50 mg/l) for the L. reuteri and LBSagar plates (KEBOLAB AB, Lund, Sweden) ) for the total Lactobacilluscount. MRS-3 is a modified MRS agar (KEBOLAB AB, Lund, Sweden)containing 2% sodium acetate (wt/vol). LBS agar is prepared asrecommended by the manufacturer by adding 1.32 ml glacial acetic acidper liter. Agar plates were incubated anaerobically using BBL Gas packsin anaerobic jars) at 37° C. for 48 hours. DNA from selected isolatesfrom the study (see below) were analyzed by PCR using a BacterialBarcodes repPROTM DNA Fingerprinting kit (Bacterial BarCodes, Inc.,Houston, Tex.), and the fingerprints were analyzed using Bionumericssoftware (Applied Maths BVBA, Sint-Martens-Latem, Belgium).

At the start of the study and before administration of the StudyProduct, none of the gastroscopy session subjects had detectable L.reuteri in the feces (Table 2). After 28 days of L. reuteri intake, allof them had L. reuteri in the feces in amounts ranging from 1.0×10² to3.5×10⁵ CFU/g (colony-forming units per gram fecal material), with amean of 4.0×10⁴ CFU/g, indicating a significant increase in live L.reuteri in the feces due to administration of the Study Product. Fivesubjects elected to deliver fecal samples on days 42 and 56 (i.e., up to28 days after administration of the Study Product). In these subjects,the L. reuteri persisted in the feces four weeks after intake (Table 2).TABLE 2 Recovery of L. reuteri in feces Gastroscopy subjects Subject 1 23 4 5 6 Tablets consumed 100 104 104 86 93 92 % compliance  89  93  9377 83 82 CFU L. reuteri/g faeces Day 0 nd Nd nd Nd nd nd Day 28 5.0E+031.0E+03 3.8E+04 3.5E+05 1.0E+02 1.0E+02 Day 42 * 1.4E+03 1.5E+03 * *1.2E+03 Day 56 * * 1.3E+03 * * 2.2E+03 Gastroscopy subjects Subject 7 89 10 Mean SD Tablets consumed 81 104 102 98 96 8 % compliance 72  93  9188 86 7 CFU L. reuteri/g faeces Day 0 nd nd nd nd nd Day 28 4.1E+032.0E+02 1.0E+02 1.9E+03 4.0E+04 1.1E+05 Day 42 1.6E+03 6.0E+02 3.0E+025.0E+02 1.0E+03 5.3E+02 Day 56 1.0E+03 6.0E+02 * 1.0E+03 1.2E+03 6.0E+02Ileoscopy subjects Subject 11 12 13 14 15 16 17 18 19 Mean SD Tabletsconsumed 92 110 112 110 98 110 112 112 112 108 7 % compliance 82  98 100 98 88  98 100 100 100  96 7 CFU L. reuteri/g ileostomy output Day 0 ndNd nd Nd nd nd nd 5.00E+04 1.00E+02 nd Day 28 nd Nd nd 6.8E+03 1.0E+023.0E+02 5.0E+02  4.0E+04  1.0E+02 8.0E+03 1.6E+04 Day 42 * 1.0E+03 *6.0E+02 1.9E+03 6.0E+02 * * * 1.0E+03 6.1E+02 Day 56 * 1.0E+03 *8.0E+02 * 2.0E+02 * * * 6.7E+02 4.2E+02nd = not detected (<1.0 × 102 CFU/g faecal material);*indicates no sample given by the subjectFecal (ileostomy output) samples were taken at recruitment (Day 0)before supplementation with L. reuteri for 28 days.Results are expressed as colony forming units (CFU)/g wet weight fecalmaterial.

EXAMPLE 5 Histology

To evaluate any local immunological response, the changes in amount ofB-lymphocytes, T-lymphocytes and macrophages were determined. Baselinebiopsies and day-28 biopsies were formalin-fixed and imbedded inparaffin. Subsequently, 4 μm sections were cut and stainedhistochemically using standard techniques (Hematoxylin-eosin, vanGieson, PeriodicAcidSciff-Alcain and PeriodicAcidSciff-diastase) andimmunohistochemically. The primary antibodies, obtained from DAKO,Glostrup, Denmark, were to: CD20 (B-lymphocytes), CD3, CD4+, CD8(T-lymphocytes), CD68 (histiocytes), Helicobacter, and Ki-67(proliferation marker). The immunohistochemical staining was performedon the DAKO TechMate™ 500 immunostainer to obtain uniform staining.

A pathologist evaluated the biopsies. On the basis of histochemicalstaining the tissue damage was graded (1-10) according to Madsen et al.(Gastroenterol. 1999; 116:1107-1114). This grade represents thenumerical sum of four criteria: ulceration, epithelial hyperplasis, theamount of mononuclear cells, and neutrophil granulocytes in the laminapropria. On the basis of the immunohistochemical staining, the number ofCD20, CD3, CD4+, CD8 and CD68 positive cells in lamina propria wereevaluated semi-quantitatively. Three months later, the baseline andday-28 biopsies were blinded and re-evaluated by the same pathologist.The overall correlation between positive stained cells of the baselineand the day-28 biopsies were calculated in each evaluation and compared.

Predominantly single B-lymphocyte cells were found in the stomach(corpus and antrum) of two subjects at day 0 and two subjects on day 28.One subject had dispersed cells in the stomach (antrum) on day 28. Foursubjects had single cells in the duodenum at day 0 and 8 subjects hadpredominantly single cells in the duodenum at day 28. Eight subjects hadpredominantly single cells of CD3, CD4+ and CD8 (T-lymphocytes) in thestomach (corpus and antrum) at day 0 and nine subjects had predominantlysingle cells in the ventricle (corpus and antrum) at day 28 (Table 3).Dispersed cells were found in the duodenum in 8 subjects at day 0 andpredominantly dispersed T-lymphocytes were found in the duodenum of 7subjects at day 28.

Predominantly single histiocyte cells were found in the stomach (corpusand antrum) in nine subjects at day 0 and in 5 subjects at day 28.Predominantly dispersed cells were found in the duodenum of 6 subjectsat day 0 and day 28 (not the same 6 subjects).

There was a statistically significant fall in the number of histiocytes(CD68) in the biopsies from the corpus (p=0.025) and antrum (p=0.046),and a significantly increased number of B-cells (CD20) in the duodenumafter subjects had received L. reuteri (p=0.046); Table 3). Nosignificant changes were found in the amount of T-lymphocytes due to theintake of L. reuteri. Concordance between the two investigations (first,not-blinded, and second, blinded analysis of histological samples) was76% (Table 3). For the histiocytes (CD68) cells of the corpus andantrum, the concordance was only 40% and 65%, respectively. Concordanceof the duodenal B-cells (CD20) was 60%. Thus, there was some uncertaintyin these findings; Concordance in the CD3, CD4+, and CD8 analyses wasmuch higher (up to 90%; Table 3).

In the ileoscopy session, all biopsies were histologically normal andHelicobacter negative. Ki-positive cells were normal in all biopsies.Predominantly single B-lymphocyte cells were found in 6 subjects anddispersed cells in 2 subjects at day 0. Single cells were observed inall subjects at day 28. Predominantly dispersed CD3 cells(B-lymphocytes) were found in 7 subjects on both day 0 and day 28.Predominantly dispersed CD4+ cells were found in 7 subjects on day 0 andpredominantly adjoining groups of cells in 7 subjects on day 28.Predominantly dispersed CD8 cells were found in 7 subjects on day 0 andin 9 subjects on day 28 (Table 3). Predominantly dispersed histiocytecells were found in 7 subjects on both day 0 and day 28.

There was a significantly higher amount of CD4+ lymphocytes aftersupplementation with L. reuteri for 28 days (p=0.046; Table 3). Nosignificant changes were found in the amount of B-lymphocytes orhistiocytes due to the administration of L. reuteri. Concordance betweenthe two investigations was generally high for all cells types examinedand for the CD4+ findings it was 78% indicating good reliability inthese data (Table 3). All Helicobacter stainings were negative. TABLE 3Histological evaluation of ileal biopsies from subjects in the ileoscopysession Day 0 Day 28 Histology B-cells T-cells Histiocyte HistologyB-cells T-cells Histiocyte Subject score CD 20 CD 3 CD4 CD8 CD68 ScoreCD 20 CD 3 CD4 CD8 CD 68 11 0 X XX XX XX XX 0 X X XXX XX XX 12 0 XX XXXXXX XX XX 0 X XX XXX XX XX 13 0 — XX XX XX XX 0 X XX XXX XX X 14 0 X XXXX XX XX 0 X X XXX XX XX 15 0 XX XXX XX XX XX 0 X XX XXX XX X 16 0 X XXXX XX XX 0 X XX XXX XX XX 17 0 X XX XX X XX 0 X XX XX XX XX 18 0 X XX XXX X 0 X XX XX XX XX 19 0 X XX XXX XX X 0 X XX XXX XX XXBiopsies were taken within the distal 20 cm of the samll bowel and werefixed and sectioned for histological Examination usingimmunohistochemical staining of specific cell types (see text).“—” = no cells detected;“X” = single cells;“XX” = dispersed cells or aggregations of cells;“XXX” = several adjoining groups of cells.Histology score (1 to 10) grading the degree of tissue damage.Statistical analysis: Wilcoxon Signed Rank Test. The samples were givena nominal score —, X, XX and XXX were set to 1, 2, 3 & 4 respectivelyfor statistical analysis.

1. Use of Lactobacillus reuteri strains that a. Exhibit good toxin binding and neutralizing effect; which may be confirmed by bringing L.reuteri cells or supernatant in contact with toxin and test for the difference in available toxin e.g. by antibody test and b. Exhibit good CD4+ cell recruitment which may be tested by using antibodies against CD4 e.g. with immunohistochemical immunoflourescent methods for the producing of a composition for improving immune-function in mammals.
 2. A product comprising a strain with at least the characteristics according to claim
 1. 3. The product of claim 2 wherein the product is formulated as a food containing cells of the selected strain.
 4. The product of claim 2 wherein the product is formulated as a tablet containing cells of the selected strain.
 5. The product of claim 2 wherein the product is formulated as a dietary supplement containing cells of the selected strain.
 6. The product of claim 2 wherein the product is formulated as a confectionery containing cells of the selected strain.
 7. The product of claim 2 wherein the product is formulated as a drug containing cells of the selected strain.
 8. The use of the culture supernatant of L. reuteri ATCC 55730 not neutralizing bacterial Toxins.
 9. A method for improving immune-function in mammals using Lactobacillus reuteri strains in products containing cells of such strains, comprising: using strains that a. Exhibit good toxin binding and neutralizing effect; which may be confirmed by bringing L.reuteri cells or supernatant in contact with toxin and test for the difference in available toxin e.g. by antibodies b. and exhibit good CD4+ cell recruitment, which may be tested by using antibodies against CD4 e.g. with immunohistochemical immunoflourescent methods. 